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    <title>Developing Performance Benchmarks</title>
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        <h1 align="center">Developing Performance Benchmarks</h1>
      	<p align="left">Originating from recognition that like for many other 
		molecular applications, lambda gDNA could be used as a universal 
		standard for qPCR, lambda-based assays provided a foundation 
		from which to develop and test LRE. This included investigations into 
		the impact of enzyme formulation, SYBR Green I concentration, reaction 
		volume, amplicon size, GC content, and cycling regime<sup><a href="#1">1</a>,<a href="#2">2</a></sup>. </p>
		<p align="left">The general utility of lambda-based performance 
		benchmarks became even more evident during broad implementation of LRE, 
		which included testing of new instruments and/or enzyme formulations, 
		in addition to becoming a central part of the quality control protocols 
		provided by the LRE Analyzer. As discussed in the
		<a href="optical_precision_overview.html">optical precision overview</a>, 
		a prominent example are differences in optical performance as reflected 
		by scattering of within the LRE plot, which 
		for the examples below were generated with 100 fg lambda gDNA + CAL1, 
		amplified with QuantiTect using an identical cycling regime (95 <sup>o</sup>C 
		10sec, 65 <sup>o</sup>C 120 sec):</p>
		<p align="center"><b>AB7500 normal ramping</b><br>
		<img border="0" src="images/lre_plot_scattering3.gif" width="260" height="149"><br>
		<b>AB7500 fast ramping<br>
		</b>
		<img border="0" src="images/7900_lre_plot.gif" width="258" height="144"><br>
		<b>Mx3000P<br>
		</b>
		<img border="0" src="images/lre_plot_scattering1.gif" width="261" height="147"></p>
		<p>Particularly for cycles within the lower region of the amplification 
		plots, it is clear that the Applied Biosystems 7500 with normal ramping 
		produces the highest optical precision, as reflected by high level of 
		conformity to the LRE line. It is thus recommended that a standard calibration reaction set 
		consisting of 100 fg lambda gDNA and CAL1 be included during 
		implementation of LRE, in order to assess the general performance of 
		your instrument and reaction setup.</p>
		<p>Other examples include <a href="monitoring_assay_performance.html">
		monitoring assay performance</a> and identification of anomalies 
		produced by the enzyme formulation such as
		<a href="../issues/profile_arcing.html">profile arcing</a>. </p>
		<p>This is a table relating lambda gDNA mass to the number of genomes </p>
		<p align="center">
		<img border="0" src="images/lambda_table.gif" width="170" height="204"></p>
		<p align="left"><a name="1">1</a>.
		Rutledge RG, Stewart D (2008) A kinetic-based sigmoidal model for the 
		polymerase chain reaction and its application to high-capacity absolute 
		quantitative real-time PCR. BMC Biotechnol 8: 47</p>
		<p align="left"><a name="2">2</a>.
		Rutledge RG, Stewart D (2010) Assessing the performance capabilities of 
		LRE-based assays for absolute quantitative real-time PCR. PLoS ONE 5: 
		e9731.</p>
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